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A microfluidics-based stem cell model of early post-implantation human development

Abstract

Early post-implantation human embryonic development has been challenging to study due to both technical limitations and ethical restrictions. Proper modeling of the process is important for infertility and toxicology research. Here we provide details of the design and implementation of a microfluidic device that can be used to model human embryo development. The microfluidic human embryo model is established from human pluripotent stem cells (hPSCs), and the resulting structures exhibit molecular and cellular features resembling the progressive development of the early post-implantation human embryo. The compartmentalized configuration of the microfluidic device allows the formation of spherical hPSC clusters in prescribed locations in the device, enabling the two opposite regions of each hPSC cluster to be exposed to two different exogenous chemical environments. Under such asymmetrical chemical conditions, several early post-implantation human embryo developmental landmarks, including lumenogenesis of the epiblast and the resultant pro-amniotic cavity, formation of a bipolar embryonic sac, and specification of primordial germ cells and gastrulating cells (or mesendoderm cells), can be robustly recapitulated using the microfluidic device. The microfluidic human embryo model is compatible with high-throughput studies, live imaging, immunofluorescence staining, fluorescent in situ hybridization, and single-cell sequencing. This protocol takes ~5 d to complete, including microfluidic device fabrication (2 d), cell seeding (1 d), and progressive development of the microfluidic model until gastrulation-like events occur (1–2 d).

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Fig. 1: Design and preparation of the microfluidic device and scheme of cell loading to establish initial cell clusters.
Fig. 2: Microscopy images showing examples of problems that might be encountered while implementing the protocol.
Fig. 3: Microfluidic generations of epiblast-like cyst, posteriorized embryonic-like sac (P-ELS), and anteriorized embryonic-like sac (A-ELS).
Fig. 4: Posteriorized and anteriorized gastrulating cell development in the microfluidic device.

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Data availability

Representative results obtained using this protocol are available within the article, with additional examples available from the corresponding author upon request.

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Acknowledgements

This work is supported by the University of Michigan Mechanical Engineering Faculty Support Fund, the Michigan-Cambridge Research Initiative, and the University of Michigan Mcubed Fund. The Lurie Nanofabrication Facility at the University of Michigan is acknowledged for support with microfabrication.

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Authors and Affiliations

Authors

Contributions

Y.Z. and J.F. conceived and initiated the project; Y.Z. designed, performed, and quantified the experiments; Y.S. helped to design experiments; Y.Z. and J.F. wrote the manuscript; J.F. supervised the study. All authors edited and approved the manuscript.

Corresponding author

Correspondence to Jianping Fu.

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Competing interests

Y.Z., Y.S., and J.F. have filed two provisional patents related to this work (US provisional patent application nos. 62/431,907 and 62/897,565).

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Peer review information Nature Protocols thanks Guohao Dai, Eric Siggia, and Patrick Tam for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Key references using this protocol

Zheng, Y. et al. Nature 573, 421–425 (2019): https://doi.org/10.1038/s41586-019-1535-2

Shao, Y. et al. Nat. Mater. 16, 419–425 (2017): https://doi.org/10.1038/nmat4829

Shao, Y. et al. Nat. Commun. 8, 208 (2017): https://doi.org/10.1038/s41467-017-00236-w

Supplementary information

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Supplementary Data 1

Zipped file containing a CAD file for the photomask used for microfabrication of the microfluidic device and a pdf of the photomask design.

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Zheng, Y., Shao, Y. & Fu, J. A microfluidics-based stem cell model of early post-implantation human development. Nat Protoc 16, 309–326 (2021). https://doi.org/10.1038/s41596-020-00417-w

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